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Differential gene expression during capillary morphogenesis in 3D collagen matrices

regulated expression of genes involved in basement membrane matrix assembly, cell cycle progression, cellular differentiation and G-protein signaling

Department of Pathology and Laboratory Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA

View larger version (66K): [in a new window]   Fig. 1. Capillary morphogenesis in 3D collagen matrices. Human ECs were suspended in 3D collagen matrices and, at varying times of cultures, were fixed, stained with toluidine blue and photographed. Some cultures were embedded in plastic and thin sectioned to obtain cross-sectional views of the capillary tubes. Times of the analysis are indicated. Arrowheads show ECs with intracellular vacuoles and arrows indicate an EC-lined capillary tube. Bar, 50 µm (8 hour panel); bar, 50 µm (120 hours panel).

View larger version (20K): [in a new window]   Fig. 2. Patterns of gene expression observed during capillary morphogenesis in 3D collagen matrices. Five different major patterns of gene expression were observed in this system, indicated as patterns A-E. Genes, whose expression patterns most closely fit these patterns, are indicated next to each pattern.

View larger version (51K): [in a new window]   Fig. 3. Differential expression of genes during capillary morphogenesis as determined by reverse transcriptase-PCR and northern blot analyses. Total RNA was prepared from EC cultures at 0, 8, 24 and 48 hours of culture. The RNA was reverse transcribed and PCR was performed using gene-specific primer sets (left panel). Control G3PDH is shown to indicate its stable expression and that equal loading occurred throughout the lanes. Selected genes were analyzed for expression using northern blot analysis (right panel). 3 µg of total RNA was loaded per lane for each time point and blots were probed with 32P-labelled cDNAs. In the upper panels, the upper arrow indicates the position of the 28S rRNA subunit, whereas the lower arrow indicates the position of the 18S rRNA subunit. The lower panels show just the area of the blot with the hybridizing band. The G3PDH blot indicates equal loading of RNA and stable expression of G3PDH mRNA during the time course.

View larger version (60K): [in a new window]   Fig. 4. Differential gene expression during capillary morphogenesis as determined by western blot analysis and immunohistochemistry. EC-collagen gel extracts or conditioned medium were prepared or collected at the indicated time points. Samples were run on SDS-PAGE and, after blotting, were probed with antibodies directed to various proteins. Cell extracts were probed with antibodies directed to collagen type IV, 2-macroglobulin and Id-1. Conditioned medium samples were probed with antibodies directed to collagen type IV or laminin. The arrows in the collagen type IV blots represent the 1 and 2 chains of collagen type IV. Arrows indicate the position of molecular weight markers on the blots. In B, a 48 hour culture was stained with antibodies to collagen type IV under non-permeabilizing conditions. No staining was observed without the addition of the primary anti-collagen type IV antibody. Bar, 50 µm.

View larger version (40K): [in a new window]   Fig. 5. Differential expression of extracellular matrix and integrin genes during capillary morphogenesis. RT-PCR was performed to assess the mRNA expression patterns of various extracellular matrix proteins (A) and integrin subunits (B) over a 48 hour time course. Total RNA was prepared from EC cultures at 0, 8, 24 and 48 hours of culture. The RNA was reverse transcribed for RT-PCR and PCR was performed using gene specific primer sets.

View larger version (35K): [in a new window]   Fig. 6. Interference with collagen type IV synthesis and secretion blocks capillary morphogenesis in 3D collagen matrices. EC cultures were treated with or without ethyl 3,4-dihydroxybenzoate (edb), an inhibitor of prolyl hydoxylase. Cultures were fixed at varying times, stained, quantitated for lumen formation (B) and photographed (A). Bar, 50 µm (A). Cell extract samples (from cultures with or without edb at 300 µM) and at different times of culture were collected and run on 7% SDS-PAGE gels (C). Lanes 1 and 2, 0 hours; lanes 3,4, 8 hours; lanes 5,6, 24 hours; lanes 7,8, 48 hours; lanes 9,10, 72 hours. Proteins were blotted to PVDF membranes and were probed with antibodies to collagen type IV. Arrows indicate the position of the 1 and 2 chains of collagen type IV.

View larger version (35K): [in a new window]   Fig. 7. Differential expression of novel capillary morphogenesis genes (CMGs) during morphogenesis in 3D collagen matrices. Differential display and cDNA library screening were used to identify differentially expressed genes during capillary morphogenesis. DNA sequencing revealed whether genes were known or novel and gene-specific primers were prepared to assess differential expression by RT-PCR (A). RT-PCR was performed to assess the mRNA expression patterns of various CMGs and known genes over a 48 hour time course. Total RNA was prepared from EC cultures at 0, 8, 24 and 48 hours of culture. The RNA was reverse transcribed for RT-PCR and PCR was performed using gene-specific primer sets. In addition, cDNA probes were made in select cases to perform northern blots (B). The far-left panel in B represents a methylene blue-stained blot indicating the position of the 28S and 18S rRNA bands and showing equal loading of 3 µg of total RNA per lane. (C) A western blot of cell extracts from a time course of capillary morphogenesis is shown using affinity purified CMG-2 antibodies. MCH, melanin concentrating hormone; GCPKRK, germinal center protein kinase related kinase-1.

View larger version (50K): [in a new window]   Fig. 8. Amino acid sequences, domain analyses and mRNA tissue distribution of two novel differentially expressed genes, CMG-1 and CMG-2. (A) Amino acid sequence of CMG-1. Bold letters indicates a coiled-coil domain structure and underlined letters indicate potential tyrosine phosphorylation sites. The nucleotide sequence of CMG-1 has been submitted to GenBank (accession no. AY040325). (B) Amino acid sequence of CMG-2. The bold letters proximal to the transmembrane segment show homology to a von Willebrand Factor A domain and the putative transmembrane domain is underlined. The nucleotide sequence of CMG-2 has been submitted to GenBank (accession no. AY040326). (C) RT-PCR analysis of mRNA tissue (adult versus fetal) distribution of CMG-1 and CMG-2. The tissue distribution of control G3PDH is also shown.

View larger version (43K): [in a new window]   Fig. 9. Intracellular targeting of CMG-1-GFP and CMG-2-GFP fusion proteins. CMG-1-GFP and CMG-2-GFP fusion protein vectors were created by preparing recombinant adenoviral vectors encoding the fusion proteins. A control GFP-only virus was also constructed. Recombinant adenoviruses were infected into 293 cells (a-d) or endothelial cells (e-g). In a-c, individual cells were photographed 24 hours after infection with CMG-1-GFP virus. Arrowheads represent fluorescent vesicular structures. In d, individual cells were photographed 24 hours after infection with GFP only virus. Bar, 20 µm (a-d). In e-g, endothelial cells were infected with CMG-2-GFP virus for 24 hours and were fixed and permeabilized for immunofluorescence staining. Panel e shows cells expressing the CMG-2-GFP protein. Panel f shows cells stained with anti-Hsp47 antibodies followed by staining with rhodamine-conjugated rabbit anti-mouse antibodies. No background staining was observed by the addition of the secondary antibodies alone. In g, a double exposure of the same field photographed in e and f is shown, illustrating the colocalization of CMG-2-GFP and Hsp47. Bar, 20 µm (e-g).

View larger version (19K): [in a new window]   Fig. 10. Recombinant CMG-2 binds to the basement membrane matrix proteins, collagen type IV and laminin. The 20 kDa domain proximal (residues 34-214) to the putative transmembrane domain was expressed as a histidine-tagged fusion protein in E.coli and purified by Ni/Cd-sepharose chromatography. Five µg of the protein was run on a 12% SDS-PAGE gel and was stained by Coomassie blue (A). A schematic diagram of CMG-2 is shown with the von Willebrand Factor A domain homology noted from residues 43-212 which is within the 20 kDa recombinantly produced protein (B). A control protein, histidine-tagged GFP, was similarly purified as a control. These proteins were absorbed to plastic at 10 µg/ml and after blocking with detergent, were incubated with 1 µg/ml of biotinylated collagen type IV, laminin, osteopontin, fibronectin and human serum albumin in 0.1% Tween-20 in Tris-buffered saline, pH 7.5. After a 1 hour incubation at 25°C, the wells were washed and then incubated with 1 µg/ml of avidin-peroxidase in the same Tween-20-Tris buffer for 30 minutes. The wells were then washed, developed for peroxidase activity and the plate read in an ELISA plate reader at 490 nm. The values shown are derived from triplicate wells + s.d. (C).